A N N A L E S UNIVERSITATIS MARIAE CURIE-SKŁODOWSKA LUBLIN – POLONIA VOL. LXIII, 4 SECTIO AA 2008 Biomedical applications of the Langmuir monolayer technique ♣ K. H ąc-Wydro and P. Dynarowicz-Łątka Jagiellonian University, Faculty of Chemistry, Ingardena 3, 30-060 Kraków, Poland The modeling of natural membranes is a consequence of their complex structure. The paper describes different approaches used to model biomembranes, and emphasizes the advantages of applying the Langmuir monolayer technique in this aspect. 1. INTRODUCTION The technique of monomolecular layers formed at the aqueous solution-air interface, termed Langmuir (or insoluble, spread, floating) monolayers (films) is based on spreading an aliquot of an amphiphile of interest in organic, volatile and water-immiscible solvent (such as chloroform) on water surface. After solvent evaporation, the free surface is entirely covered by a monomolecular layer of an amphiphile, which can be compressed to the desired surface pressure/mean molecular area by sliding barriers, using the Langmuir trough [1]. The use of the Langmuir method allows for a continuous control of both quality of the surface and such parameters as molecular packing, physical state, lateral pressure and composition. A thorough understanding of monolayer behavior at the free water surface is essential for exploiting so-called Langmuir-Blodgett (LB) technique [2], which basically involves the formation of a monolayer film on water with its subsequent transfer (either by vertical or horizontal dipping) onto a solid substrate, as a viable route for making highly ordered, defectless ultrathin films with controllable molecular orientation, thickness and architecture. These outstanding opportunities of the LB method have led to an ♣ Paper dedicated to Professor Emil Chibowski on the occasion of his 65 th birthday 48 K. H ąc-Wydro and P. Dynarowicz-Łątka international effort to exploit such films in optical devices, highly specific chemical sensors or molecular electronics. It is quite understandable that the application of Langmuir films, because of the fact that they are formed on water, is much more limited as compared to LB films. However, there is a field, in which they are very important, i.e. biomedical sciences, like biology, pharmacy and medicine. Lipids monolayers form an excellent model of one leaflet of a cellular membrane and therefore the Langmuir technique is successfully applied to studying the properties of biomembranes, various processes occurring on membrane level or the interactions between membrane components. Such a technique is also useful tool for investigating of the mechanism of action of amphiphilic drugs active on membrane level or the effect of other biomolecules on biomembrane. This paper is aimed at providing examples for successful applications of the Langmuir technique in this area. 2. MODELING OF A CELLULAR MEMBRANE The natural membrane is composed of different class of lipids (mainly phospholipids, sphingolipids and sterols) and proteins organized into the structure described generally by so-called fluid-mosaic model proposed by Singer and Nicolson in 1972 [3,4]. The framework of membrane builds a lipid bilayer, which is a “fluid” part of this structure. The “mosaic”, on the other hand, is made by proteins embedded into the lipids’ framework. Proteins either penetrate the bilayer (integral proteins) or are localized on the surface of leaflets. The latter may be located loosely on the surface layer (peripheral proteins) or bound covalently to membrane lipids (lipid-anchored proteins). The fluid-mosaic model represents general structure of a biomembrane; however, its organization is still being investigated. For example, the research performed in last decades proved the existence of microdomains enriched in cholesterol, called “lipid rafts” [5]. This finding significantly changed previously accepted view on homogeneous distribution of lipids in natural membranes. The cellular membrane is characterized by a highly dynamic structure, in which both phospholipids and proteins are mobile and able to interact. Membrane is also asymmetric in structure – this means that the composition of the two membrane layers is different. Generally, in the outer layer phosphatidylcholines and sphingomyelins are mainly present, while the inner layer contains phosphatidylethanolamines and phosphatidylserines [4]. Obviously, the composition of a membrane (lipids type and the lipid-to-protein proportion) depends on the species of organism, kind of an organ and tissue, type of a cell or, within the cell, on the type of organelle. Biomedical applications of the Langmuir monolayer technique 49 The cellular membrane is not only a physical barrier separating the inside of the cell from the outside, but allows cells to selectively interact with their environment [4,6]. Apart from its importance in vast array of cellular processes (such as ions and metabolites transport, communication and regulation processes), it makes a site of a number of drugs acting at the membrane level of a living cell. Antimicrobial peptides, such as alamethicin [7] or gramicidin [8], polyene macrolide antibiotics (for example amphotericin B or nystatin [9]) or alkyl-lysophospholipids (a new generation anticancer drugs, like miltefosine [10] or edelfosine [11]) are good examples of molecules, the physiological activity of which occurs at a lipid membrane interface. For these particular kinds of drugs, studies of their influence on the cell membranes are of utmost importance. For this purpose, several approaches are possible. The drug-membrane interactions can be investigated with living cells or natural membranes isolated from cells. However, the foregoing methods are characterized by complexity of both the experimental procedures and the results obtained, and moreover, provide only global information on the membrane. Therefore they are unsuitable for studying specific aspects of a given phenomena occurring at membrane level (e.g. lipid – protein or drug – lipid interactions). For these kinds of investigation the best choice is to use one of membrane models. They are briefly discussed below. glycolipid proteins phospholipid bilayer sterol Fig. 1. Schematic representation of a biomembrane structure, according to the fluid- mosaic model. The most widely used membrane models are lipid vesicles (liposomes), which consist of an aqueous space closed in a lipid bilayer(s) (Figure 2). Depending on the number of layers surrounding the aqueous core the liposomes are classified as multillamelar vesicles and unilamellar vesicles [12,13]. These structures were discovered in the sixties of the preceding century upon microscopic observation of phospholipids dispersion in water [12,13]. Originally 50 K. H ąc-Wydro and P. Dynarowicz-Łątka prepared liposomes were formed from natural phospholipids. Later, other amphiphatic compounds were found to be useful for making liposomes and artificial vesicles were prepared from synthetic amphiphiles. Currently, depending either on the type of an amphiphile used or on the application of vesicles, the liposomes are specifically named (niosomes, nanoparticles, nanospheres) [12,13]. To distinguish the artificial liposomes formed by surfactants from those prepared from natural phospholipids, the former are called “vesicles ”, while the name “ liposomes ” is reserved for the latter systems [14]. Fig. 2. Scheme of a lipid vesicle. Liposomes formed from phospholipids can successfully mimic the dynamic, fluid and semi-permeable natural bilayer[15]. Therefore, vesicles – in general- are widely used to study the properties of various type of membranes, cellular processes (e.g. endo- and exocytosis, cell lysis, transport phenomena) or protein- membrane lipid interactions [16,17], effect of biomolecules on phospholipid bilayer [18-20] or the interaction of drugs with membrane components [21-23]. Although initially liposomes were used mainly to model the natural membranes, with time their application has significantly broadened and now they are also applied in medicine and pharmacy (drug delivery, preparation of less toxic drug formulations [24,25]), in medical diagnostics, gene therapy, cosmetics, food- industry, or in the environmental protection [26-28]. Although liposomes are easy to prepare and allow various spectroscopic measurements, they suffer from several limitations. Firstly, the range over which the lipid concentration can be varied without changing the surface curvature and physical state is limited. It is not possible to regulate lipid lateral packing density and lipid composition independently. Moreover, the physical state of compositionally identical vesicles depends on the method of preparation. It is also difficult to prepare a homogeneous (in size and layer number) vesicle Biomedical applications of the Langmuir monolayer technique 51 suspension and to avoid a spontaneous fusion. Another severe disadvantage is a small curvature radius that imposes strong constraints at the polar head level. Monomolecular (Langmuir) films formed at the free surface of aqueous solutions overcome all the above limitations as it has already been mentioned in the Introduction . In addition, with the Langmuir technique contrary to liposomes, it is possible to mimic similar conditions as in cellular membrane. It was found that the pressure in biological membranes corresponds to the surface pressure of 30- 35 mN/m in the Langmuir experiment [29]. The use of monolayers as a membrane model system will be discussed separately